Retinopathy of prematurity (ROP) is a leading cause of childhood blindness world-wide. Our lab studies two important sequential phases of ROP: Phase I, in which physiologic retinal vascular development (PRVD) is delayed; followed by Phase II, in which vaso-proliferative intra-vitreal neovascularization (IVNV) increases risk of blindness. Standard-of-care laser treatment and anti-angiogenic strategies intended to treat Phase II, such as inhibitors of vascular endothelial growth factor (VEGF), are destructive of developing retinal tissue or delay PRVD, thereby prolonging Phase I; treatments intended to reduce the delay period of Phase I and to advance PRVD, can worsen IVNV in Phase II. Clinically, one wishes to inhibit IVNV but not interfere with PRVD in developing preterm infants. Work in the previous grant period identified targets to inhibit IVNV safely using a rat model of ROP and led to the following mechanistic hypotheses: VEGF produced by Muller cells (MCs) promotes survival in photoreceptors, retinal neurons, and MCs, but also binds VEGF receptor 2 in endothelial cells (ECs) to activate the erythropoietin receptor (EPOR) and/or NOX4/NADPH oxidase. Interactions between VEGFR2 and EPOR or NOX4 exacerbate EC-STAT3, which causes phase II IVNV. We also developed a method to study molecular mechanisms in the rat model of ROP using lentiviral gene therapy and polymerase II promoters targeting specifically MCs or ECs, which drive shRNAs efficiently when embedded in microRNA30 (miR30). Specific Aim 1 is to test if knockdown of VEGF164 in MCs to retinal VEGF levels that inhibit IVNV and not delay PRVD will allow retinal neuronal survival and function. Specific Aim 2 is to test if knockdown of EPOR in ECs will reduce IVNV in phase II ROP and not delay PRVD in phase I. Specific Aim 3 is to test if STAT3 knockdown in ECs will inhibit IVNV and not delay PRVD. We will also test whether a drug treatment to regulate NOX4/VEGFR2-mediated STAT3 activation can inhibit phase II IVNV. Methods include: lentiviral gene therapy techniques; oxygen-induced retinopathy models in rat (50/10 ROP) and in transgenic mice; Micron III imaging; sub retinal injections; optical coherence tomography; electroretinography; immunohistochemistry of flat mounts and sections; western blot, real-time PCR.